Anticoagulant agents

ABSTRACT

This invention relates to a compound of the Formula I 
     
         X-C(O)--Y-G-R                                              I 
    
     (wherein X, Y, G and R have the values defined in the description), or a pharmaceutically acceptable salt thereof, processes and intermediates for the preparation of such a compound or salt, pharmaceutical compositions comprising such a compound or salt and methods of their use as thrombin inhibitors, coagulation inhibitors and agents for the treatment of thromboembolic disorders.

This application is a divisional of application Ser. No. 08/879,637,filed Jun. 20,1997, now U.S. Pat. No. 5,863,929 the entire disclosure ofwhich herein is incorprated by reference, which application claims thebenefit of U.S. Provisional Application No. 60/020,371, filed Jun. 25,1996.

This invention relates to thrombin inhibitors which are usefulanticoagulants in mammals. In particular it relates toortho-hydroxybenzamidine derivatives having high anticoagulant activity.Thus, this invention relates to new inhibitors of thrombin,pharmaceutical compositions containing the compounds as activeingredients, and the use of the compounds as anticoagulants forprophylaxis and treatment of thromboembolic disorders such as venousthrombosis, pulmonary embolism, arterial thrombosis, in particularmyocardial ischemia, myocardial infarction and cerebral thrombosis,general hypercoagulable states and local hypercoagulable states, such asfollowing angioplasty and coronary bypass operations, and generalizedtissue injury as it relates to the inflammatory process. In addition,the agents are useful as anticoagulants in in vitro applications.

The process of blood coagulation, thrombosis, is triggered by a complexproteolytic cascade leading to the formation of thrombin. Thrombinproteolytically removes activation peptides from the Aα-chains and theBβ-chains of fibrinogen, which is soluble in blood plasma, initiatinginsoluble fibrin formation.

Anticoagulation is currently achieved by the administration of heparinsand coumarins. Parenteral pharmacological control of coagulation andthrombosis is based on inhibition of thrombin through the use ofheparins. Heparins act indirectly on thrombin by accelerating theinhibitory effect of endogenous antithrombin III (the main physiologicalinhibitor of thrombin). Because antithrombin III levels vary in plasmaand because surface-bound thrombin seems resistant to this indirectmechanism, heparins can be an ineffective treatment. Because coagulationassays are believed to be associated with efficacy and with safety,heparin levels must be monitored with coagulation assays (particularlythe activated partial thromboplastin time (APTT) assay). Coumarinsimpede the generation of thrombin by blocking the posttranslationalgamma-carboxylation in the synthesis of prothrombin and other proteinsof this type. Because of their mechanism of action, the effect ofcoumarins can only develop slowly, 6-24 hours after administration.Further, they are not selective anticoagulants. Coumarins also requiremonitoring with coagulation assays (particularly the prothrombin time(PT) assay).

Recently, interest has grown in small synthetic molecules whichdemonstrate potent direct inhibition of thrombin. See, for exampleRobert M. Scarborough, Annual Reports in Medicinal Chemistry, (1995),30, 71-80, where inhibitors which lack a polarizable functionality tointeract with the active site Ser-195 hydroxy group of thrombin aretermed active site inhibitors. Active site inhibitors in which theC-terminal moiety comprises an unsubstituted or certain substitutedamidinophenyl (benzamidine) moiety are exemplified in EP 623596, WO94/29336, WO 95/23609 and WO 95/35309. The amidinophenyl moiety isstrongly basic, a property which militates against good oralbioavailability. See, for example R. J. Misra, et al., Bioorganic &Medicinal Chemistry Letters, (1994), 4, 2165-2170, where less basicargatroban analogs were shown to retain useful thrombin inhibitorypotency while exhibiting better distribution properties as shown byenhanced Caco-2 cell permeability. As discussed below, the compoundsdisclosed herein retain useful thrombin inhibitory potency whileexhibiting improved distribution coefficients as a result of theirparticularly substituted amidinophenyl moieties. Subsequent to thepriority date for the instant application, there were publishedinternational patent applications WO 96/24609 and WO 96/25426 disclosingcertain substituted amidinophenyl compounds, includingD-cyclohexylglycyl-N-[[4-(aminoiminomethyl)-3-hydroxyphenyl]methyl-L-prolinamidedihydrochloride at Example 53 of WO 96/25426.

Although the heparins and coumarins are effective anticoagulants, nogenerally accepted commercial drug has yet emerged from the smallsynthetic molecules; and despite the continuing promise for this classof compounds, there still exists a need for anticoagulants which actselectively on thrombin, and which, independent of antithrombin III,exert inhibitory action shortly after administration, preferably by anoral route, and do not interfere with lysis of blood clots, as requiredto maintain hemostasis.

The present invention is directed to the discovery that the compounds ofthe present invention, as defined below, are potent thrombin inhibitorsthat may have high bioavailability following oral administration.

According to the invention there is provided a compound having theFormulaI

    X-C(O)--Y-G-R                                              I

wherein

X-C(O)-- is D-prolinyl, D-homoprolinyl, R^(m) -(CH₂)_(g) --NH--CH₂--C(O)--, ##STR1## in which R^(d) is carboxy or methylsulfonyl;

R^(e) is NHR^(c), NHCOR^(c) or NHCOOR^(c) ; in which

R^(c) is (C₁ -C₁₀)alkyl, (C₃ -C₈)cycloalkyl or a (C₃ -C₈)cycloalkyl-(C₁-C₆)alkyl radical of 4-10 carbons;

T is (C₃ -C₈)cycloalkyl, (C₁ -C₈)alkyl, ##STR2## a is 0, 1 or 2; and Qis --OH, (C₁ -C₄)alkoxy, or --NH--A;

A is hydrogen, (C₁ -C₄)alkyl, R"SO₂ --, R"OC(O)--, R"C(O)--, R^(n)C(O)-- or --(CH₂)_(g) --R^(m) ;

g is 1, 2, or 3;

B is hydrogen or (C₁ -C₄)alkyl;

R' is hydrogen or (C₁ -C₄)alkyl;

R" is (C₁ -C₄)alkyl, (C₁ -C₄)fluoroalkyl bearing one to five fluoros,--(CH₂)_(d) --R^(m), or unsubstituted or substituted aryl, where aryl isphenyl, naphthyl, a 5- or 6-membered unsubstituted or substitutedaromatic heterocyclic ring, having one or two heteroatoms which are thesame or different and which are selected from sulfur, oxygen andnitrogen, or a 9- or 10-membered unsubstituted or substituted fusedbicyclic aromatic heterocyclic group having one or two heteroatoms whichare the same or different and which are selected from sulfur, oxygen andnitrogen;

R^(m) is --COOR^(b), --SO₂ (C₁ -C₄ alkyl), --SO₃ H, --P(O) (OR^(b))₂ ortetrazol-5-yl;

R^(n) is --COOR^(b) or tetrazol-5-yl;

each R^(b) is independently hydrogen or (C₁ -C₄)alkyl;

d is 1, 2, or 3;

m is 0, 1, or 2;

n is 0, 1, or 2; and

Z is hydrogen, (C₁ -C₄)alkyl, (C₁ -C₄)alkoxy, hydroxy, halo or (C₁-C₄)alkylsulfonylamino;

--Y--G-- is ##STR3## in which r is 0, 1 or 2;

R^(g) is (C₁ -C₆)alkyl, (C₃ -C₈)cycloalkyl, or --(CH₂)_(p)--L--(CH₂)_(q) --T';

R^(p) is (C₁ -C₆)alkyl, (C₃ -C₈)cycloalkyl, or --(CH₂)_(p)--L--(CH₂)_(q) --T';

where p is 0, 1, 2, 3, or 4; L is a bond, --O--, --S--, or --NH--; q is0, 1, 2 or 3; and T' is (C₁ -C₄)alkyl, (C₃ -C₈)cycloalkyl, --COOH,--CONH₂, or Ar, where Ar is unsubstituted or substituted aryl, wherearyl is phenyl, naphthyl, a 5- or 6-membered unsubstituted orsubstituted aromatic heterocyclic ring, having one or two heteroatomswhich are the same or different and which are selected from sulfur,oxygen and nitrogen, or a 9- or 10-membered unsubstituted or substitutedfused bicyclic aromatic heterocyclic group having one or two heteroatomswhich are the same or different and which are selected from sulfur,oxygen and nitrogen;

R^(y) is --CH₂ --, --O--, --S--, or --NH--; and

R^(z) is a bond or, when taken with R^(y) and the three adjoining carbonatoms, forms a saturated carbocyclic ring of 5-8 atoms, one atom ofwhich may be --O--, --S--, or --NH--;

--G--R is --C(O)--NH--(CH₂)_(s) --R, --CH₂ --NH--(CH₂)_(s) --R, --CH₂--NH--C(O)--R or --(CH₂)_(t) --O--R in which s is 1 or 2 and t is 1, 2or 3; and

R is a 4-amidino-3-hydroxyphenyl group bearing 0, 1, 2 or 3 fluorosubstituents;

or a pharmaceutically acceptable salt thereof.

In addition to a compound of Formula I, the present invention provides apharmaceutical composition comprising a compound of Formula I, or apharmaceutically acceptable salt thereof, in association with apharmaceutically acceptable carrier, diluent or excipient.

The present invention further provides a method of inhibiting thrombincomprising administering to a mammal in need of treatment, a thrombininhibiting dose of a compound of Formula I.

The present invention also provides a method of inhibiting thrombosis ina mammal comprising administering to a mammal in need of treatment, anantithrombotic dose of a compound of Formula I.

This invention relates to new inhibitors of thrombin, pharmaceuticalcompositions containing the compounds as active ingredients, and the useof the compounds as anticoagulants for prophylaxis and treatment ofthromboembolic diseases such as venous thrombosis, pulmonary embolism,arterial thrombosis, in particular myocardial ischemia, myocardialinfarction and cerebral thrombosis, general hypercoagulable states andlocal hypercoagulable states, such as following angioplasty and coronarybypass operations, and generalized tissue injury as it relates to theinflammatory process.

In this specification, the following definitions are used, unlessotherwise described: Halo is fluoro, chloro, bromo or iodo. Alkyl,alkoxy, etc. denote both straight and branched groups; but reference toan individual radical such as "propyl" embraces only the straight chain("normal") radical, a branched chain isomer such as "isopropyl" beingspecifically denoted.

The term "5- or 6-membered aromatic heterocyclic ring" means any 5- or6-membered ring that will afford a stable structure containing one ortwo nitrogen atoms; one sulfur atom; one oxygen atom; one nitrogen andone sulfur atom; or one nitrogen and one oxygen atom. The 5-memberedring has two double bonds and the 6-membered ring has three doublebonds.

The term "9- or 10-membered fused bicyclic aromatic heterocyclic group"means any bicyclic group in which any of the above 5- or 6-memberedrings is ortho fused to a benzene ring or to a 6-membered heterocyclicaromatic ring as defined above in a manner that will afford a stablestructure.

It will be appreciated that many of the above heterocycles may exist intautomeric forms. All such forms are included within the scope of thisinvention.

Each of the aromatic or heteroaromatic groups listed for the definitionof Ar or R" is independently unsubstituted or substituted with one ortwo substituents that will afford a stable structure independentlyselected from halo, hydroxy, (C₁ -C₄)alkyl, (C₁ -C₄)alkoxy, amino,mono(C₁ -C₄ alkyl)amino, di(C₁ -C₄ alkyl)amino, --(CH₂)_(j) COOH,mercapto, --S(O)_(h) (C₁ -C₄ alkyl), --NHS(O)_(h) (C₁ -C₄ alkyl),--NHC(O) (C₁ -C₄ alkyl), --S(O)_(h) NH₂, --S(O)_(h) NH(C₁ -C₄ alkyl), or--S(O)_(h) N(C₁ -C₄ alkyl)₂, h is 0, 1 or 2, and j is 0, 1, 2, 3, or 4.

In the representation of Formula I, the carbonyl functionality of groupX--(CO)-- is attached to the amine functionality of the --Y-- group.

The group ##STR4## where Z and A are both hydrogen, is referred to attimes herein as phenylglycyl and abbreviated Phg. Compounds wherein Ais, e.g., methyl, are referred to as the N.sup.α -methyl-phenylglycylgroup and abbreviated MePhg. Substituted compounds wherein Z is otherthan hydrogen are referred to by the type and position of thesubstituent group, e.g., 3'-chlorophenylglycyl or Phg(3-Cl). The group##STR5## where Z and A are both hydrogen, is referred to at times hereinas phenylalanyl and abbreviated Phe. Compounds wherein A is, e.g.,methyl, are referred to as the N.sup.α -methyl-phenylalanyl group andabbreviated MePhe. Substituted compounds wherein Z is other thanhydrogen are referred to by the type and position of the substituentgroup, e.g., 3'-chlorophenylalanyl or Phe(3-Cl).

The groups ##STR6## when R' is hydrogen, are referred to at times hereinas 1- and 3-tetrahydro-isoquinolinecarbonyl, respectively, and arerespectively abbreviated 1-Tiq and 3-Tiq.

The groups ##STR7## when R' is hydrogen, are referred to at times hereinas 1- and 3-perhydro-isoquinolinecarbonyl, respectively, and arerespectively abbreviated 1-Piq and 3-Piq. As indicated by the crookedlines, various ring fusion isomers of these substituents exist--thisinvention contemplates any individual isomer and combinations thereof.

The group ##STR8## wherein r is 0, 1, or 2 is referred to asazetidine-2-carbonyl, prolinyl, or homoprolinyl, and is abbreviated Azt,Pro or hPro, respectively.

The group ##STR9## represents a saturated bicyclic system of the 4,5;5,5; 6,5; 7,5; or 8,5 type. The stereochemistry at 3a is cis to thecarbonyl; the other bridgehead bond may be either cis or trans exceptthat the 4,5 and 5,5 systems must be cis at the bridgehead. Thedefinitions of R^(y) and R^(z) provide that the variable ring, whichincludes the three carbon atoms shown, is a saturated carbocyclic systemof 4-8 atoms. All of the ring atoms may be carbon, or one of the ringatoms may be a hetero atom selected from --O--, --S--, and --NH--. Thisdefinition includes the moiety derived from octahydroindole-2-carboxylicacid, as represented by ##STR10## The various cis and trans forms ofthis moiety are contemplated by this invention. The preferred isomerderived from [2S-(2α,3αβ,7αβ)]-octahydro-indole-2-carboxylic acid isabbreviated "Ohi" and is represented by ##STR11##

The asterisks in radical Y denote a chiral center that corresponds to(L) in the natural amino acids. The asterisk in radical X denotes achiral center that is (D) or (DL); the # in radical X denotes a chiralcenter that is (L)

It will be appreciated that certain compounds of Formula I may exist in,and be isolated in, isomeric forms, including tautomeric forms or cis-or trans-isomers, as well as optically active racemic or diastereomericforms. The present invention encompasses a compound of Formula I in anyof the tautomeric forms or as a mixture thereof. It is to be understoodthat the present invention encompasses a compound of Formula I as amixture of diastereomers, as well as in the form of an individualdiastereomer, and that the present invention encompasses a compound ofFormula I as a mixture of enantiomers, as well as in the form of anindividual enantiomer, any of which mixtures or form possessesinhibitory properties against thrombin, it being well known in the arthow to prepare or isolate particular forms and how to determineinhibitory properties against thrombin by standard tests including thosedescribed below.

In addition, a compound of Formula I may exhibit polymorphism or mayform a solvate with water or an organic solvent. The present inventionalso encompasses any such polymorphic form, any solvate or any mixturethereof.

Particular values are listed below for radicals (either alone or as partof another radical), substituents, and ranges, for illustration only,and they do not exclude other defined values or other values withindefined ranges for the radicals and substituents.

A particular value for a (C₁ -C₄)alkyl group, a (C₁ -C₆)alkyl group, a(C₁ -C₈)alkyl group or a (C₁ -C₁₀)alkyl group is methyl, ethyl, propyl,isopropyl, butyl, isobutyl or t-butyl. A particular value for a (C₁-C₄)alkoxy group is methoxy, ethoxy, propoxy, isopropoxy, or t-butyloxy.A particular value for a (C₃ -C₈)cycloalkyl group is cyclopropyl,cyclopentyl or cyclohexyl. A particular value for a (C₁ -C₄)fluoroalkylgroup is trifluoromethyl or 2,2,2-trifluoroethyl. A particular value foraryl is phenyl, naphthyl, furyl, thienyl, pyridyl, indolyl, quinolinylor isoquinolinyl.

A particular compound of Formula I as defined above is one in which

X--C(O)-- is D-homoprolinyl, ##STR12## in which T is cyclohexyl orphenyl; a is 0 or 1; and A is hydrogen, (C₁ -C₄)alkyl, (C₁ -C₄alkyl)sulfonyl, (C₁ -C₄ alkyl)-oxy-carbonyl, (C₁ -C₄ alkyl)carbonyl orcarboxymethyl; and

--Y--G-- is --NR^(g) --CH₂ --G--, ##STR13## in which R^(g) is (C₁-C₆)alkyl, --(CH₂)_(q) --(C₃ -C₈)cycloalkyl or --(CH₂)_(q) -phenyl; q is0, 1, 2 or 3; and r is 0, 1, or 2;

or a pharmaceutically acceptable salt thereof.

A preferred compound of Formula I as defined above is one in which##STR14## wherein T is cyclohexyl; a is 1; and A is hydrogen,ethylsulfonyl or carboxymethyl, particularly carboxymethyl; and

--Y--G-- is ##STR15## in which r is 0 or 1; or a pharmaceuticallyacceptable salt thereof.

For any of the above defined compounds of Formula I, a particular valueof --G--R is --C(O)--NH--(CH₂)_(s) --R; and a preferred value of --G--Ris --C(O)--NH--(CH₂)_(s) --R in which s is 1, i.e. --C(O)--NH--CH₂ --R.

A particular compound of Formula I in which --G--R is --C(O)--NH--CH₂--R and the other groups have any of the above definitions may bedenoted by Formula Ia ##STR16## in which f is 0, 1, 2 or 3.

For any of the above defined compounds of Formula I, a particular valuefor R is 4-amidino-3-hydroxyphenyl or4-amidino-3-hydroxy-2,5,6-trifluorophenyl; and a more particular valueis 4-amidino-3-hydroxyphenyl.

A particular compound of the invention is one of those described hereinas Example 1, 2, 3, 4, 5, 9, 10 or 11; and a preferred compound is onedescribed as Example 1, 3 or 5, particularly Example 3; or apharmaceutically acceptable salt thereof.

A compound of Formula I may be made by processes which include processesknown in the chemical art for the production of structurally analogouscompounds or by a novel process described herein. Novel processes andintermediates for the manufacture of a compound of Formula I as definedabove provide further features of the invention and are illustrated bythe following procedures in which the meanings of the generic radicalsare as defined above, unless otherwise specified. It will be recognizedthat it may be preferred or necessary to prepare a compound of Formula Iin which a functional group is protected using a conventional protectinggroup, then to remove the protecting group to provide the compound ofFormula I. (A) For a compound of Formula I in which --G--R is--C(O)--NH--(CH₂)_(s) --R, coupling an acid of Formula II,

    X--C(O)--Y--C(O)--OH                                       II

or an activated derivative thereof, with an arine of Formula III.

    H.sub.2 N--(CH.sub.2).sub.s --R                            III

The coupling is carried out using a conventional procedure, for exampleby using a coupling reagent such asbenzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, forexample as described in Example 1-E, or such as1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, for example as describedin Example 5.

(B) Coupling an acid of Formula IV,

    X--C(O)--OH                                                IV

or an activated derivative thereof, with an amine of Formula V.

    H--Y--G--R                                                 V

The coupling is carried out using a conventional procedure, such as byusing one of the methods described above in (A).

(C) Hydrogenolyzing the N--O bond of a corresponding compound of FormulaVI ##STR17## in which f is 0, 1, 2 or 3. Conveniently, thehydrogenolysis is carried out using a palladium on carbon catalyst inacidic, aqueous alchohol at ambient temperature and under hydrogen atambient or a few bars' pressure; and the product is isolated as its acidaddition salt.

Whereafter, for any of the above procedures, when a functional group isprotected using a protecting group, removing the protecting group.

Whereafter, for any of the above procedures, when a pharmaceuticallyacceptable salt of a compound of Formula I is required, it is obtainedby reacting the acidic or basic form of such a compound of Formula Iwith a base or an acid affording a physiologically acceptable counterionor by any other conventional procedure, such as, for example, exchangingthe counterion of a salt.

A compound corresponding to compound of Formula I in which one or morefunctional groups is protected provides another aspect of the invention.Such a compound may be represented as a compound of Formula Ip

    (P.sup.X)X--C(O)--(P.sup.Y)Y--G(P.sup.G)--R(P.sup.R)       Ip

which bears one or more of the protecting groups P^(X), P^(Y), P^(G) andP^(R) wherein P^(X) is an optional protecting group(s) for a functionalgroup(s) of X--C(O)--; P^(Y) is an optional protecting group(s) for afunctional group(s) of --Y--; P^(G) is an optional amino protectinggroup for G when G--R is --(CH₂)--NH--(CH₂)_(s) --R; and P^(R) is anoptional protecting group(s) for a functional group of R. Typical valuesfor P^(X) and P^(Y) include the groups which form a t-butylester orbenzyl ester when the protected functional group is carboxy, the groupswhich form a t-butyl urethane or a benzyl urethane when the protectedfunctional group is amino, and the groups which form a methyl ether,t-butyl ether or benzyl ether when the protected functional group ishydroxy. It will be recognized that some compounds of Formula I mayserve as a protected equivalent of another compound of Formula I. Forexample, a compound of Formula I in which A is R"OC(O)-- wherein R" ist-butyl is a protected equivalent of a compound of Formula I in which Ais hydrogen, as described in Example 1. Similarly, a compound of FormulaI in which R^(m) is --COOR^(b) wherein R^(b) t-butyl is a protectedequivalent of a compound of Formula I in which R^(m) is --COOR^(b) andR^(b) is hydrogen.

As mentioned above, the invention includes a pharmaceutically acceptablesalt of a thrombin inhibiting compound defined by the above Formula I. Aparticular benzamidine of this invention possesses one or moresufficiently basic functional groups to react with any of a number ofnontoxic inorganic and organic acids to form a pharmaceuticallyacceptable salt. Acids commonly employed to form acid addition salts areinorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid, phosphoric acid, and the like, and organic acidssuch as p-toluene sulfonic, methanesulfonic acid, oxalic acid, p-bromophenyl sulfonic acid, carbonic acid, succinic acid, citric acid, benzoicacid, acetic acid, and the like. Examples of such pharmaceuticallyacceptable salts thus are the sulfate, pyrosulfate, bisulfate, sulfite,bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate,metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate,propionate, decanoate, caprylate, acrylate, formate, isobutyrate,caproate, heptanoate, propiolate, oxalate, malonate, succinate,suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate,xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, gamma-hydroxybutyrate, glycollate, tartrate,methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate, and the like. Preferredpharmaceutically acceptable acid addition salts include those formedwith mineral acids such as hydrochloric acid, hydrobromic acid andsulfuric acid.

For a compound of Formula I in which X or Y bears an acidic moiety, sucha a carboxy group, a pharmaceutically acceptable salt may be made with abase which affords a pharmaceutically acceptable cation, which includesalkali metal salts (especially sodium and potassium), alkaline earthmetal salts (especially calcium and magnesium), aluminum salts andammonium salts, as well as salts made from physiologically acceptableorganic bases such as triethylamine, morpholine, piperidine andtriethanolamine.

If not commercially available, the necessary starting materials for thepreparation of a compound of Formula I may be prepared by procedureswhich are selected from standard techniques of organic chemistry,including aromatic and heteroaromatic substitution and transformation,from techniques which are analogous to the syntheses of known,structurally similar compounds, especially peptide syntheses, andtechniques which are analogous to the above described procedures orprocedures described in the Examples. It will be clear to one skilled inthe art that a variety of sequences is available for the preparation ofthe starting materials. Starting materials and procedures which arenovel provide further aspects of the invention.

A starting material acid of Formula II also may be represented as anacid of Formula IIp

    (P.sup.X)X--C(O)--(P.sup.Y)Y--C(O)--OH                     IIp

in which P^(X) and P^(Y) are optional protecting groups as definedabove. Conveniently, an acid of Formula IIp may be prepared by couplingan optionally protected acid of Formula VII

    (P.sup.X)X--C(O)--OH                                       VII

with an amino acid derivative of Formula VIII

    H--(P.sup.Y)Y--C(O)--OP.sup.C                              VIII

in which P^(C) is hydrogen or a carboxy protecting group, such as forexample methyl, ethyl, t-butyl or benzyl, followed by removal of theprotecting group P^(C), when present.

A convenient general route for the preparation of an amine of FormulaIII or an amine of Formula V is outlined in Scheme I, in which G^(a)represents a latent or protected form of the group H₂ N--(CH₂)_(s) -- orthe group H--Y--G--, respectively, and f is 0, 1, 2 or 3. ##STR18##

Thus, according to the method of Shutske and Kapples (J. HeterocyclicChem. (1989), 26, 1293-1298), an ortho-fluoro benzonitrile of Formula Xis treated with the potassium anion of acetone oxime to afford thecorresponding oxime of Formula XI; acid hydrolysis of the oxime affordsthe amine of Formula XII which cyclizes in situ to afford thesubstituted 3-amino-1,2-benzisoxazole derivative of Formula XIII. Thegroup G^(a) may be converted into H₂ N--(CH₂)_(s) -- to afford an amineof Formula XIV or into H--Y--G to afford an amine of Formula XV,respectively; hydrogenolysis of the benzisoxazole, using a proceduresimilar to that described in (C) above, then affords the respectiveamine of Formula III or Formula V. Alternatively, it may be preferred tofirst hydrogenolyze the benzisoxazole of Formula XIII to a correspondingcompound of Formula XVI before transforming the group G^(a) to afford anamine of Formula III or Formula V. As described at Example 1-D and atExample 2-B, the conversion of G^(a) (as cyano) into H₂ N--CH₂ -- may beperformed at the same time as the hydrogenolysis, thus providing a"one-pot" conversion of a compound of Formula XIII into an amine ofFormula III.

A starting material of Formula VI may be prepared by a route analogousto one described above, for example by using a compound of Formula XIVor Formula XV, or a protected derivative thereof.

A compound of the invention is isolated best in the form of an acidaddition salt. A salt of the compound of Formula I formed with an acidsuch as one of those mentioned above is useful as a pharmaceuticallyacceptable salt for administration of the antithrombotic agent and forpreparation of a formulation of the agent. Other acid addition salts maybe prepared and used in the isolation and purification of the compound.

One of the novel intermediates of the invention is a compound of FormulaIII, or a salt and/or protected derivative thereof. A particularcompound of Formula III is one in which s is 1 and which may berepresented by Formula IIIa ##STR19## in which f is 0, 1, 2 or 3. Aparticular compound of Formula IIIa is one in which f is 0 or 3.

An additional aspect of the invention is the use of a compound ofFormula III (or Formula IIIa) as defined above, or a salt or protectedderivative thereof, as a starting material in the synthesis of athrombin inhibitor.

As another aspect of the invention, there is provided a novel structuralfragment of the formula ##STR20## wherein f is 0, 1, 2 or 3(particularly f is 0 or 3) as a novel structural element in a thrombininhibitor, particularly in a peptidomimetic thrombin inhibitor.

Another novel intermediate of the invention is a compound of FormulaXIII in which Ga is cyano and which may be represented by Formula XIIIa##STR21## wherein f is 0, 1, 2 or 3; particularly wherein f is 0 or 3.

As noted above, the optically active isomers and diastereomers of thecompounds of Formula I are also considered part of this invention. Suchoptically active isomers may be prepared from their respective opticallyactive precursors by the procedures described above, or by resolving theracemic mixtures. This resolution can be carried out by derivatizationwith a chiral reagent followed by chromatography or by repeatedcrystallization. Removal of the chiral auxiliary by standard methodsaffords substantially optically pure isomers of the compounds of thepresent invention or their precursors. Further details regardingresolutions can be obtained in Jacques, et al., Enantiomers, Racemates,and Resolutions, John Wiley & Sons, 1981.

The compounds of the invention are believed to selectively inhibitthrombin over other proteinases and nonenzyme proteins involved in bloodcoagulation without appreciable interference with the body's naturalclot lysing ability (the compounds have a low inhibitory effect onfibrinolysis). Also, they generally exhibit increased selectivity forthrombin compound to the prior amidinophenyl compounds. Further, suchselectivity is believed to permit use with thrombolytic agents withoutsubstantial interference with thrombolysis and fibrinolysis.

The invention in one of its aspects provides a method of inhibitingthrombin in mammals comprising administering to a mammal in need oftreatment an effective (thrombin inhibiting) dose of a compound ofFormula I.

In another of its aspects, the invention provides a method of treating athromboembolic disorder comprising administering to a mammal in need oftreatment an effective (thromboembolic disorder therapeutic and/orprophylactic amount) dose of a compound of Formula I.

The invention in another of its aspects provides a method of inhibitingcoagulation in a mammal comprising administering to a mammal in need oftreatment an effective (coagulation inhibiting) dose of a compound ofFormula I.

The thrombin inhibition, coagulation inhibition and thromboembolicdisorder treatment contemplated by the present method includes bothmedical therapeutic and/or prophylactic treatment as appropriate.

In a further embodiment the invention relates to treatment, in a humanor other mammal, of conditions where inhibition of thrombin is required.The compounds of the invention are expected to be useful in mammals,including man, in treatment or prophylaxis of thrombosis andhypercoagulability in blood and tissues. Disorders in which thecompounds have a potential utility are in treatment or prophylaxis ofthrombosis and hypercoagulability in blood and tissues. Disorders inwhich the compounds have a potential utility, in treatment and/orprophylaxis, include venous thrombosis and pulmonary embolism, arterialthrombosis, such as in myocardial ischemia, myocardial infarction,unstable angina, thrombosis-based stroke and peripheral arterialthrombosis. Further, the compounds have expected utility in thetreatment or prophylaxis of atherosclerotic disorders (diseases) such ascoronary arterial disease, cerebral arterial disease and peripheralarterial disease. Further, the compounds are expected to be usefultogether with thrombolytics in myocardial infarction. Further, thecompounds have expected utility in prophylaxis for reocclusion afterthrombolysis, percutaneous transluminal angioplasty (PTCA) and coronarybypass operations. Further, the compounds have expected utility inprevention of rethrombosis after microsurgery. Further, the compoundsare expected to be useful in anticoagulant treatment in connection withartificial organs and cardiac valves. Further, the compounds haveexpected utility in anticoagulant treatment in hemodialysis anddisseminated intravascular coagulation. A further expected utility is inrinsing of catheters and mechanical devices used in patients in vivo,and as an anticoagulant for preservation of blood, plasma and otherblood products in vitro. Still further, the compounds have expectedutility in other diseases where blood coagulation could be a fundamentalcontributing process or a source of secondary pathology, such as cancer,including metastasis, inflammatory diseases, including arthritis, anddiabetes. The anti-coagulant compound is administered orally,parenterally e.g. by intravenous infusion (iv), intramuscular injection(im) or subcutaneously (sc).

The specific dose of a compound administered according to this inventionto obtain therapeutic and/or prophylactic effects will, of course, bedetermined by the particular circumstances surrounding the case,including, for example, the compound administered, the rate ofadministration, the route of administration, and the condition beingtreated.

A typical daily dose for each of the above utilities is between about0.01 mg/kg and about 1000 mg/kg. The dose regimen may vary e.g. forprophylactic use a single daily dose may be administered or multipledoses such as 3 or 5 times daily may be appropriate. In critical caresituations a compound of the invention is administered by iv infusion ata rate between about 0.01 mg/kg/h and about 20 mg/kg/h and preferablybetween about 0.1 mg/kg/h and about 5 mg/kg/h.

The method of this invention also is practiced in conjunction with aclot lysing agent e.g. tissue plasminogen activator (t-PA), modifiedt-PA, streptokinase or urokinase. In cases when clot formation hasoccurred and an artery or vein is blocked, either partially or totally,a clot lysing agent is usually employed. A compound of the invention canbe administered prior to or along with the lysing agent or subsequent toits use, and preferably further is administered along with aspirin toprevent the reoccurrence of clot formation.

The method of this invention is also practiced in conjunction with aplatelet glycoprotein receptor (IIb/IIa) antagonist, that inhibitsplatelet aggregation. A compound of the invention can be administeredprior to or along with the IIb/IIIa antagonist or subsequent to its useto prevent the occurrence or reoccurrence of clot formation.

The method of this invention is also practiced in conjunction withaspirin. A compound of the invention can be administered prior to oralong with aspirin or subsequent to its use to prevent the occurrence orreoccurrence of clot formation. As stated above, preferably a compoundof the present invention is administered in conjunction with a clotlysing agent and aspirin.

This invention also provides pharmaceutical compositions for use in theabove described therapeutic method. Pharmaceutical compositions of theinvention comprise an effective thrombin inhibiting amount of a compoundof formula I in association with a pharmaceutically acceptable carrier,excipient or diluent. For oral administration the antithromboticcompound is formulated in gelatin capsules or tablets which may containexcipients such as binders, lubricants, disintegration agents and thelike. For parenteral administration the antithrombotic is formulated ina pharmaceutically acceptable diluent e.g. physiological saline (0.9percent), 5 percent dextrose, Ringer's solution and the like.

The compound of the present invention can be formulated in unit dosageformulations comprising a dose between about 0.1 mg and about 1000 mg.Preferably the compound is in the form of a pharmaceutically acceptablesalt such as for example the sulfate salt, acetate salt or a phosphatesalt. An example of a unit dosage formulation comprises 5 mg of acompound of the present invention as a pharmaceutically acceptable saltin a 10 ml sterile glass ampoule. Another example of a unit dosageformulation comprises about 10 mg of a compound of the present inventionas a pharmaceutically acceptable salt in 20 ml of isotonic salinecontained in a sterile ampoule.

The compounds can be administered by a variety of routes including oral,rectal, transdermal, subcutaneous, intravenous, intramuscular, andintranasal. The compounds of the present invention are preferablyformulated prior to administration. Another embodiment of the presentinvention is a pharmaceutical composition comprising an effective amountof a compound of Formula I or a pharmaceutically acceptable salt orsolvate thereof in association with a pharmaceutically acceptablecarrier, diluent or excipient therefor.

The active ingredient in such formulations comprises from 0.1 percent to99.9 percent by weight of the formulation. By "pharmaceuticallyacceptable" it is meant the carrier, diluent or excipient must becompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

The present pharmaceutical compositions are prepared by known proceduresusing well known and readily available ingredients. The compositions ofthis invention may be formulated so as to provide quick, sustained, ordelayed release of the active ingredient after administration to thepatient by employing procedures well known in the art. In making thecompositions of the present invention, the active ingredient willusually be admixed with a carrier, or diluted by a carrier, or enclosedwithin a carrier which may be in the form of a capsule, sachet, paper orother container. When the carrier serves as a diluent, it may be asolid, semi-solid or liquid material which acts as a vehicle, excipientor medium for the active ingredient. Thus, the compositions can be inthe form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols, (as asolid or in a liquid medium), soft and hard gelatin capsules,suppositories, sterile injectable solutions, sterile packaged powders,and the like.

The following formulation examples are illustrative only and are notintended to limit the scope of the invention in any way. "Activeingredient," of course, means a compound according to Formula I or apharmaceutically acceptable salt or solvate thereof.

Formulation 1: Hard gelatin capsules are prepared using the followingingredients:

    ______________________________________                                                      Quantity                                                          (mg/capsule)                                                                ______________________________________                                        Active ingredient                                                                             250                                                             Starch, dried 200                                                             Magnesium stearate 10                                                         Total 460 mg                                                                ______________________________________                                    

Formulation 2: A tablet is prepared using the ingredients below:

    ______________________________________                                                        Quantity                                                        (mg/tablet)                                                                 ______________________________________                                        Active ingredient 250                                                           Cellulose, microcrystalline 400                                               Silicon dioxide, fumed 10                                                     Stearic acid 5                                                                Total 665 mg                                                                ______________________________________                                    

The components are blended and compressed to form tablets each weighing665 mg.

Formulation 3: An aerosol solution is prepared containing the followingcomponents:

    ______________________________________                                                            Weight                                                    ______________________________________                                        Active ingredient     0.25                                                      Ethanol 25.75                                                                 Propellant 22 (Chlorodifluoromethane) 70.00                                   Total 100.00                                                                ______________________________________                                    

The active compound is mixed with ethanol and the mixture added to aportion of the propellant 22, cooled to -30° C. and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remainder of the propellant. The valveunits are then fitted to the container.

Formulation 4: Tablets, each containing 60 mg of active ingredient, aremade as follows:

    ______________________________________                                        Active ingredient        60 mg                                                  Starch 45 mg                                                                  Microcrystalline cellulose 35 mg                                              Polyvinylpyrrolidone (as 10% solution in water)  4 mg                         Sodium carboxymethyl starch 4.5 mg                                            Magnesium stearate 0.5 mg                                                     Talc  1 mg                                                                    Total 150 mg                                                                ______________________________________                                    

The active ingredient, starch and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The aqueous solution containingpolyvinylpyrrolidone is mixed with the resultant powder, and the mixturethen is passed through a No. 14 mesh U.S. sieve. The granules soproduced are dried at 50° C. and passed through a No. 18 mesh U.S.Sieve. The sodium carboxymethyl starch, magnesium stearate and talc,previously passed through a No. 60 mesh U.S. sieve, are then added tothe granules which, after mixing, are compressed on a tablet machine toyield tablets each weighing 150 mg.

Formulation 5: Capsules, each containing 80 mg of active ingredient, aremade as follows:

    ______________________________________                                        Active ingredient  80 mg                                                        Starch 59 mg                                                                  Microcrystalline cellulose 59 mg                                              Magnesium stearate  2 mg                                                      Total 200 mg                                                                ______________________________________                                    

The active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 45 mesh U.S. sieve, and filled into hardgelatin capsules in 200 mg quantities.

Formulation 6: Suppositories, each containing 225 mg of activeingredient, are made as follows:

    ______________________________________                                        Active ingredient    225 mg                                                     Saturated fatty acid glycerides 2,000 mg                                      Total 2,225 mg                                                              ______________________________________                                    

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

Formulation 7: Suspensions, each containing 50 mg of active ingredientper 5 ml dose, are made as follows:

    ______________________________________                                        Active ingredient   50        mg                                                Sodium carboxymethyl cellulose 50 mg                                          Syrup 1.25 ml                                                                 Benzoic acid solution 0.10 ml                                                 Flavor q.v.                                                                   Color q.v.                                                                    Purified water to total 5 ml                                                ______________________________________                                    

The active ingredient is passed through a No. 45 mesh U.S. sieve andmixed with the sodium carboxymethyl cellulose and syrup to form a smoothpaste. The benzoic acid solution, flavor and color are diluted with aportion of the water and added, with stirring. Sufficient water is thenadded to produce the required volume.

Formulation 8: An intravenous formulation may be prepared as follows:

    ______________________________________                                        Active ingredient       100 mg                                                  Isotonic saline 1,000 ml                                                    ______________________________________                                    

The solution of the above ingredients generally is administeredintravenously to a subject at a rate of 1 ml per minute.

The ability of a compound of the present invention to be an effectiveand orally active thrombin inhibitor is evaluated in one or more of thefollowing assays.

The compounds provided by the invention (Formula I) selectively inhibitthe action of thrombin in mammals. The inhibition of thrombin isdemonstrated by in vitro inhibition of the amidase activity of thrombinas measured in an assay in which thrombin hydrolyzes the chromogenicsubstrate, N-benzoyl-L-phenylalanyl-L-valyl-L-arginyl-p-nitroanilide,N-benzoyl-L-Phe-L-Val-L-Arg-p-nitroanilide.

The assay is carried out by mixing 50 μl buffer (0.03 M Tris, 0.15 MNaCl, pH 7.4) with 25 μl of human thrombin solution (purified humanthrombin, Enzyme Research Laboratories, South Bend, Ind., at 8 NIHunits/ml) and 25 μl of test compound in a solvent (50% aqueous methanol(v:v)). Then 150 μl of an aqueous solution of the chromogenic substate(at 0.25 mg/ml) are added and the rates of hydrolysis of the substrateare measured by monitoring the reactions at 405 nm for the release ofp-nitroaniline. Standard curves are constructed by plotting freethrombin concentration against hydrolysis rate. The hydrolysis ratesobserved with test compounds are then converted to "free thrombin"values in the respective assays by use of the standard curves. The boundthrombin (bound to test compound) is calculated by subtracting theamount of free thrombin observed in each assay from the known initialamount of thrombin used in the assay. The amount of free inhibitor ineach assay is calculated by subtracting the number of moles of boundthrombin from the number of moles of added inhibitor (test compound).

The Kass value is the hypothetical equilibrium constant for the reactionbetween thrombin and the test compound (I). ##EQU1##

Kass is calculated for a range of concentrations of test compounds andthe mean value reported in units of liter per mole. In general, athrombin inhibiting compound of Formula I of the instant inventionexhibits a Kass of 0.1×10⁶ L/mole or much greater. For example, each ofthe particularly preferred examples of the invention listed above wasdetermined to have a Kass of at least 100×10⁶ L/mole. Thus, thecompounds of Examples 1, 3 and 5 were found to have a Kass of 770×10⁶L/mole, 1,200×10⁶ L/mole and 100×10⁶ L/mole, respectively.

By substantially following the procedures described above for humanthrombin, and using other human blood coagulation system serineproteases and using fibrinolytic system serine proteases, with theappropriate chromogenic substrates, identified below, the selectivity ofthe compounds of the present invention with respect to the coagulationfactor serine proteases and to the fibronolytic serine proteases areevaluated as well as their substantial lack of interference with humanplasma clot fibrinolysis.

Human factors X, Xa, IXa, XIa, and XIIa are purchased from EnzymeResearch Laboratories, South Bend, Ind.; human urokinase from LeoPharmaceuticals, Denmark; and recombinant activated Protein C (aPC) isprepared at Eli Lilly and Co. substantially according to U.S. Pat. No.4,981,952. Chromogenic substrates:N-Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (for factor Xa);N-Cbz-D-Arg-Gly-Arg-p-nitroanilide (for factor IXa assay as the factorXa substrate); Pyroglutamyl-Pro-Arg-p-nitroanilide (for Factor XIa andfor aPC); H-D-Pro-Phe-Arg-p-nitroanilide (for factor XIIa); andPyroglutamyl-Gly-Arg-p-nitroanilide (for urokinase); are purchased fromKabi Vitrum, Stockholm, Sweden, or from Midwest Biotech, Fishers, Ind.Bovine trypsin is purchased from Worthington Biochemicals, Freehold,N.J., and human plasma kallikrein from Kabi Vitrum, Stockholm, Sweden.Chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide for plasmakallikrein is purchased from Kabi Vitrum, Stockholm, Sweden.N-Benzoyl-Phe-Val-Arg-p-nitroanilide, the substrate for human thrombinand for trypsin, is synthesized according to procedures described abovefor the compounds of the present invention, using known methods ofpeptide coupling from commercially available reactants, or purchasedfrom Midwest Biotech, Fishers, Ind.

Human plasmin is purchased from Boehringer Mannheim, Indianapolis, Ind.;nt-PA is purchased as single chain activity reference from AmericanDiagnostica, Greenwich, Conn.; modified-t-PA6 (mt-PA6) is prepared atEli Lilly and Company by procedure known in the art (See, Burck, et al.,J. Biol. Chem., 265, 5120-5177 (1990). Plasmin chromogenic substrateH-D-Val-Leu-Lys-p-nitroanilide and tissue plasminogen activator (t-PA)substrate H-D-Ile-Pro-Arg-p-nitroanilide are purchased from Kabi Vitrum,Stockholm, Sweden.

In the chromogenic substrates described above the three-letter symbolsIle, Glu, Gly, Pro, Arg, Phe, Val, Leu and Lys are used to indicate thecorresponding amino acid group isoleucine, glutamic acid, glycine,proline, arginine, phenylalanine, valine, leucine and lysine,respectively.

Thrombin inhibitors preferably should spare fibrinolysis induced byurokinase, tissue plasminogen activator (t-PA) and steptokinase. Thiswould be important to the therapeutic use of such agents as an adjunctto streptokinase, t-PA or urokinase thrombolytic therapy and to the useof such agents as an endogenous fibrinolysis-sparing (with respect tot-PA and urokinase) antithrombotic agents. In addition to the lack ofinterference with the amidase activity of the fibrinolytic proteases,such fibrinolytic system sparing can be studied by the use of humanplasma clots and their lysis by the respective fibrinolytic plasminogenactivators.

Materials

Dog plasma is obtained from conscious mixed-breed hounds (either sexHazelton-LRE, Kalamazoo, Mich., U.S.A.) by venipuncture into 3.8 percentcitrate. Fibrinogen is prepared from fresh dog plasma and humanfibrinogen is prepared from in-date ACD human blood at the fraction I-2according to previous procedures and specifications. Smith, Biochem. J.,185, 1-11 (1980); and Smith, et al., Biochemistry, 11, 2958-2967,(1972). Human fibrinogen (98 percent pure/plasmin free) is from AmericanDiagnostica, Greenwich, Conn. Radiolabeling of fibrinogen I-2preparations is performed as previously reported. Smith, et al.,Biochemistry, 11, 2958-2967, (1972). Urokinase is purchased form LeoPharmaceuticals, Denmark, as 2200 Ploug units/vial. Streptokinase ispurchased from Hoechst-Roussel Pharmaceuticals, Somerville, N.J.

Methods--Effects on Lysis of Human Plasma Clots by t-PA

Human plasma clots are formed in micro test tubes by adding 50 ulthrombin (73 NIH unit/ml) to 100 ul human plasma which contains 0.0229uCi 125-iodine labeled fibrinogen. Clot lysis is studied by overlayingthe clots with 50 ul of urokinase or streptokinase (50, 100, or 1000unit/ml) and incubating for 20 hours at room temperature. Afterincubation the tubes are centrifuged in a Beckman Microfuge. 25 ul ofsupernate is added into 1.0 ml volume of 0.03 M tris/0.15 M NaCl bufferfor gamma counting. Counting controls 100 percent lysis are obtained byomitting thrombin (and substituting buffer). The thrombin inhibitors areevaluated for possible interference with fibrinolysis by including thecompounds in the overlay solutions at 1, 5, and 10 ug/ml concentrations.Rough approximations of IC₅₀ values are estimated by linearextrapolations from data points to a value which would represent 50percent of lysis for that particular concentration of fibrinolyticagent.

Anticoagulant Activity

Materials

Dog plasma and rat plasma are obtained from conscious mixed-breed hounds(either sex, hazelton-LRE, Kalamazoo, Mich., U.S.A.) or fromanesthetized male Sprague-Dawley rats (Harlan Sprague-Dawley, Inc.,Indianapolis, Ind., U.S.A.) by venipuncture into 3.8 percent citrate.Fibrinogen is prepared from in-date ACD human blood as the fraction I-2according to previous procedures and specifications. Smith, Biochem. J.,185, 1-11 (1980); and Smith, et al., Biochemistry, 11, 2958-2967 (1972).Human fibrinogen is also purchased as 98 percent pure/plasmin free fromAmerican Diagnostica, Greenwich, Conn. Coagulation reagents ACTIN,Thromboplastin, and Human plasma are from Baxter Healthcare Corp., DadeDivision, Miami, Fla. Bovine thrombin from Parke-Davis (Detroit, Mich.)is used for coagulation assays in plasma.

Methods

Anticoagulation Determinations

Coagulation assay procedures are as previously described. Smith, et al.,Thrombosis Research, 50, 163-174 (1988). A CoAScreener coagulationinstrument (American LABor, Inc.) is used for all coagulation assaymeasurements. The thrombin time (TT) is measured by adding 0.05 mlsaline and 0.05 ml thrombin (10 NIH units/ml) to 0.05 ml test plasma.The activated partial thromboplastin time (APTT) is measured byincubation of 0.05 ml test plasma with 0.05 ml Actin reagent for 120seconds followed by 0.05 ml CaCl₂ (0.02 M). The prothrombin time (PT) ismeasured by adding 0.05 ml saline and 0.05 ml Thromboplastin-C reagentto 0.05 ml test plasma. The compounds of formula I are added to human oranimal plasma over a wide range of concentrations to determineprolongation effects on the TT, APTT and PT assays. Linearextrapolations are performed to estimate the concentrations required todouble the clotting time for each assay. Each of the particularlypreferred examples of the invention listed above was determined to havea TT value of less than 50 ng/mL. For example, the respective values (inng/mL) for TT were 6, 6 and 23 for the compounds of Examples 1, 3 and 5.

Animals

Male Sprague Dawley rats (350-425 gm, Harlan Sprague Dawley Inc.,Indianapolis, Ind.) are anesthetized with xylazine (20 mg/kg, s.c.) andketamine (120 mg/kg, s.c.) and maintained on a heated water blanket (37°C.). The jugular vein(s) is cannulated to allow for infusions.

Arterio-Venous Shunt Model

The left jugular vein and right carotid artery are cannulated with 20 cmlengths of polyethylene PE 60 tubing. A 6 cm center section of largertubing (PE 190) with a cotton thread (5 cm) in the lumen, is frictionfitted between the longer sections to complete the arterio-venous shuntcircuit. Blood is circulated through the shunt for 15 min before thethread is carefully removed and weighed. The weight of a wet thread issubtracted from the total weight of the thread and thrombus (see J. R.Smith, Br J Pharmacol, 77:29,1982).

FeCl₃ Model of Arterial Injury

The carotid arteries are isolated via a midline ventral cervicalincision. A thermocouple is placed under each artery and vesseltemperature is recorded continuously on a strip chart recorder. A cuffof tubing (0.058 ID×0.077 OD ×4 mm, Baxter Med. Grade Silicone), cutlongitudinally, is placed around each carotid directly above thethermocouple. FeCl₃ hexahydrate is dissolved in water and theconcentration (20 percent) is expressed in terms of the actual weight ofFeCl₃ only. To injure the artery and induce thrombosis, 2.85 ul ispipetted into the cuff to bathe the artery above the thermocouple probe.Arterial occlusion is indicated by a rapid drop in temperature. The timeto occlusion is reported in minutes and represents the elapsed timebetween application of FeCl₃ and the rapid drop in vessel temperature(see K. D. Kurz, Thromb. Res., 60:269,1990).

Spontaneous Thrombolysis Model

In vitro data suggests that peptide thrombin inhibitors inhibit thrombinand at higher concentration may inhibit, other serine proteases, such asplasmin and tissue plasminogen activator. To assess if the compoundsinhibit fibrinolysis in vivo, the rate of spontaneous thrombolysis isdetermined by implanting a labeled whole blood clot into the pulmonarycirculation. Rat blood (1 ml) is mixed rapidly with bovine thrombin (4IU, Parke Davis) and ¹²⁵ I human Fibrogen (5 μci, ICN), immediatelydrawn into silastic tubing and incubated at 37° C. for 1 hour. The agedthrombus is expelled from the tubing, cut into 1 cm segments, washed 3 Xin normal saline and each segment is counted in a gamma counter. Asegment with known counts is aspirated into a catheter that issubsequently implanted into the jugular vein. The catheter tip isadvanced to the vicinity of the right atrium and the clot is expelled tofloat into the pulmonary circulation. One hour after implant, the heartand lungs are harvested and counted separately. Thrombolysis isexpressed as a percentage where: ##EQU2## The fibrinolytic dissolutionof the implanted clot occurs time-dependently (see J. P. Clozel,Cardiovas. Pharmacol., 12:520, 1988).

Coagulation Parameters

Plasma thrombin time (TT) and activated partial thromboplastin time(APTT) are measured with a fibrometer. Blood is sampled from a jugularcatheter and collected in syringe containing sodium citrate (3.8percent, 1 part to 9 parts blood). To measure TT, rat plasma (0.1 ml) ismixed with saline (0.1 ml) and bovine thrombin (0.1 ml, 30 U/ml in TRISbuffer; Parke Davis) at 37° C. For APTT, plasma (0.1 ml) and APTTsolution (0.1 ml, Organon Teknika) are incubated for 5 minutes (37° C.)and CaCl₂ (0.1 ml, 0.025 M) is added to start coagulation. Assays aredone in duplicate and averaged.

Index of Bioavailability

A measure of bioactivity, plasma thrombin time (TT), serves as asubstitute for the assay of parent compound on the assumption thatincrements in TT resulted from thrombin inhibition by parent only. Thetime course of the effect of the thrombin inhibitor upon TT isdetermined after i.v bolus administration to anesthetized rats and afteroral treatment of fasted conscious rats. Due to limitations of bloodvolume and the number of points required to determine the time coursefrom time of treatment to the time when the response returns topretreatment values, two populations of rats are used. Each samplepopulation represents alternating sequential time points. The average TTover the time course is used to calculate area under the curve (AUC).The index of bioavailability is calculated by the formula shown belowand is expressed as percent relative activity.

The area under the curve (AUC) of the plasma TT time course isdetermined and adjusted for the dose. This index of bioavailability istermed " % Relative Activity" and is calculated as ##EQU3## Compounds

Compound solutions are prepared fresh daily in normal saline and areinjected as a bolus or are infused starting 15 minutes before andcontinuing throughout the experimental perturbation which is 15 minutesin the arteriovenous shunt model and 60 minutes in the FeCl₃ model ofarterial injury and in the spontaneous thrombolysis model. Bolusinjection volume is 1 ml/kg for i.v., and 5 ml/kg for p.o. and infusionvolume is 3 ml/hr.

Statistics

Results are expressed as means +/- SEM. One-way analysis of variance isused to detect statistically significant differences and then Dunnett'stest is applied to determine which means are different. Significancelevel for rejection of the null hypothesis of equal means is P<0.05.

Animals

Male dogs (Beagles; 18 months-2 years; 12-13 kg, Marshall Farms, NorthRose, N.Y. 14516) are fasted overnight and fed Purina certifiedPrescription Diet (Purina Mills, St. Louis, Mo.) 240 minutes afterdosing. Water is available ad libitum. The room temperature ismaintained between 66-74° F.; 45-50 percent relative humidity; andlighted from 0600-1800 hours.

Pharmacokinetic Model

Test compound is formulated immediately prior to dosing by dissolving insterile 0.9 percent saline to a 5 mg/ml preparation. Dogs are given asingle 2 mg/kg dose of test compound by oral gavage. Blood samples (4.5ml) are taken from the cephalic vein at 0.25, 0.5, 0.75, 1,2,3,4 and 6hours after dosing. Samples are collected in citrated Vacutainer tubesand kept on ice prior to reduction to plasma by centrifugation. Plasmasamples are analyzed by HPLC-MS. Plasma concentration of test compoundis recorded and used to calculate the pharmacokinetic parameters:elimination rate constant, Ke; total clearance, Cl_(t) ; volume ofdistribution, V_(D) ; time of maximum plasma test compoundconcentration, Tmax; maximum concentration of test compound of Tmax,Cmax; plasma half-life, t0.5; and area under the curve, A.U.C.; fractionof test compound absorbed, F.

Canine Model of Coronary Artery Thrombosis

Surgical preparation and instrumentation of the dogs are as described inJackson, et al., Circulation, 82, 930-940 (1990. Mixed-breed hounds(aged 6-7 months, either sex, Hazelton-LRE, Kalamazoo, Mich., U.S.A.)are anesthetized with sodium pentobarbital (30 mg/kg intravenously,i.v.), intubated, and ventilated with room air. Tidal volume andrespiratory rates are adjusted to maintain blood PO₂, PCO₂, and pHwithin normal limits. Subdermal needle electrodes are inserted for therecording of a lead II ECG.

The left jugular vein and common carotid artery are isolated through aleft mediolateral neck incision. Arterial blood pressure (ABP) ismeasured continuously with a precalibrated Millar transducer (model(MPC-500, Millar instruments, Houston, Tex., U.S.A.) inserted into thecarotid artery. The jugular vein is cannulated for blood sampling duringthe experiment. In addition, the femoral veins of both hindlegs arecannulated for administration of test compound.

A left thoracotomy is performed at the fifth intercostal space, and theheart is suspended in a pericardial cradle. A 1- to 2-cm segment of theleft circumflex coronary artery (LCX) is isolated proximal to the firstmajor diagonal ventricular branch. A 26-gauge needle-tipped wire anodalelectrode (Teflon-coated, 30-gauge silverplated copper wire) 3-4 mm longis inserted into the LCX and placed in contact with the intimal surfaceof the artery (confirmed at the end of the experiment). The stimulatingcircuit is completed by placing the cathode in a subcutaneous (s.c.)site. An adjustable plastic occluder is placed around the LCX, over theregion of the electrode. A precalibrated electromagnetic flow probe(Carolina Medical Electronics, King, N.C., U.S.A.) is placed around theLCX proximal to the anode for measurement of coronary blood flow (CBF).The occluder is adjusted to produce a 40-50 percent inhibition of thehyperemic blood flow response observed after 10-s mechanical occlusionof the LCX. All hemodynamic and ECG measurements are recorded andanalyzed with a data acquisition system (model M3000, ModularInstruments, Malvern, Pa. U.S.A.).

Thrombus Formation and Compound Administration Regimens

Electrolytic injury of the intima of the LCX is produced by applying100-μA direct current (DC) to the anode. The current is maintained for60 min and then discontinued whether the vessel has occluded or not.Thrombus formation proceeds spontaneously until the LCX is totallyoccluded (determined as zero CBF and an increase in the S-T segment).Compound administration is started after the occluding thrombus isallowed to age for 1 hour. A 2-hour infusion of the compounds of thepresent invention at doses of 0.5 and 1 mg/kg/hour is begunsimultaneously with an infusion of thrombolytic agent (e.g. tissueplasminogen activator, streptokinase, APSAC). Reperfusion is followedfor 3 hour after administration of test compound. Reocclusion ofcoronary arteries after successful thrombolysis is defined as zero CBFwhich persisted for ≧30 minutes.

Hematology and Template Bleeding Time Determinations

Whole blood cell counts, hemoglobin, and hematocrit values aredetermined on a 40-μl sample of citrated (3.8 percent) blood (1 partcitrate:9 parts blood) with a hematology analyzer (Cell-Dyn 900,Sequoia-Turner. Mount View, Calif., U.S.A.). Gingival template bleedingtimes are determined with a Simplate II bleeding time device (OrganonTeknika Durham, N.C., U.S.A.). The device is used to make 2 horizontalincisions in the gingiva of either the upper or lower left jaw of thedog. Each incision is 3 mm wide×2 mm deep. The incisions are made, and astopwatch is used to determine how long bleeding occurs. A cotton swabis used to soak up the blood as it oozes from the incision. Templatebleeding time is the time from incision to stoppage of bleeding.Bleeding times are taken just before administration of test compound (0min), 60 min into infusion, at conclusion of administration of the testcompound (120 min), and at the end of the experiment.

All data are analyzed by one-way analysis of variance (ANOVA) followedby Student-Neuman-Kuels post hoc t test to determine the level ofsignificance. Repeated-measures ANOVA are used to determine significantdifferences between time points during the experiments. Values aredetermined to be statistically different at least at the level ofp<0.05. All values are mean ± SEM. All studies are conducted inaccordance with the guiding principles of the American PhysiologicalSociety. Further details regarding the procedures are described inJackson, et al., J. Cardiovasc. Pharmacol., 21, 587-599 (1993).

Compared to the corresponding amidino-phenyl compounds, the compounds ofthe instant invention, in which a hydroxy group is juxtaposed ortho tothe amidino group, possess physio-chemical properties which are muchmore favorable for oral absorption. The logD (D=octanol/waterdistribution coefficient) at pH 7.4 [logD(7.4)] observed for thecompound of Example 5 [logD(7.4)=1.91] exhibits a more favorable valuethan that of the reference compound [logD(7.4)=-3.89], a change[.increment.logD(7.4)] of 5.80 log units. For the compound of Example 3[logD(7.4)=0.55], compared with the corresponding amidino phenylcompound, .increment.logD(7.4)=1.13 log units was observed.

The following Examples are provided to further describe the inventionand are not to be construed as limitations thereof.

The abbreviations used in the examples have the following meanings.

Amino acids: Azt=azetidine-2-carboxylic acid, Phe=phenylalamine,hPro=homo-proline, Pro=proline, Cha=β=cyclohexylalanine,Ohi=[2S-(2α,3αβ,7αβ)]-octahydro-indol-2-carboxylic acid, (1 R,4 aR,8aR)-1-Piq=(1 R,4 aR,8 aR)-1-perhydro-isoquinolinecarboxylate,Sar=sarcosine (N-methyl-glycine).

Anal.=elemental analysis

Boc=t-butyloxycarbonyl

Bn=benzyl

BOP-Cl=bis(2-oxo-3-oxazolidinyl)phosphinic chloride

t-Bu=t-butyl

n-BuLi=butyllithium

Cbz=benzyloxycarbonyl

18-Crown-6=1,4,7,10,13,16-hexaoxacyclooctadecane

DIBAL=diisobutylaluminum hydride

DMF=dimethylformamide

DMSO=dimethylsulfoxide

Et=ethyl

EtOAc=ethyl acetate

Et₂ O=diethyl ether

EtOH=ethanol

FAB-MS=fast atom bombardment mass spectrum

FD-MS=field desorption mass spectrum

HPLC=High Performance Liquid Chromatography

HRMS=high resolution mass spectrum

HOBT=1-hydroxybenzotriazole hydrate

i-PrOH=isopropanol

IR=Infrared Spectrum

Me=methyl

MeOH=methanol

NMR=Nuclear Magnetic Resonance

RPHPLC=Reversed Phase High Performance Liquid Chromatography

SiO₂ =silica gel

TEA=triethylamine

TFA=trifluoroacetic acid

THF=tetrahydrofuran

TLC=thin layer chromatography

Ts=tosyl (p-toluenesulfonyl)

The following parameters for preparative RPHPLC were employed: SolventA: 0.05% aqueous hydrochloric acid (1.5 mL concentrated hydrochloricacid in 3 L water); Solvent B: acetonitrile; Gradient: as defined ineach Example; Column: Vydac C₁₈ -5 cm×25 cm; Flow rate: 10 mL/minute.

Unless otherwise stated, pH adjustments and work up are with aqueousacid or base solutions. ¹ H-NMR indicates a satisfactory NMR spectrumwas obtained for the compound described. IR indicates a satisfactoryinfrared spectrum was obtained for the compound described.

EXAMPLE 1

Preparation ofD-cyclohexylalanyl-N-[[4-(aminoiminomethyl)-3-hydroxyphenyl]methyl]-L-prolinamidedihydrochloride ##STR22## A) Preparation of Boc-D-Cha-Pro-OH

A solution of Boc-D-Cha-OH (50.4 g, 185 mmol) in dichloromethane (360mL) was cooled to 0° C. and N-hydroxysuccinimide (22.3 g, 194 mmol) wasadded. Then 1,3-dicyclohexylcarbodiimide (39.0 g, 189 mmol) was added intwo portions as a solution in dichloromethane (90 mL). After stirringfor 3 h at 0° C., L-Pro-OH (27.6 g, 240 mmol) andN,N-diisopropylethylamine (30.9 g, 239 mmol) were added. After stirringan additional 3 h between 0° C. and 10° C., the mixture was filteredover diatomaceous earth. The filter cake was rinsed with dichloromethane(100 mL); then the combined filtrates were concentrated in vacuo. Theresidual oil was partitioned between ethyl acetate (100 mL) and 0.625 Macueous NaHCO₃ (320 mL). The layers were separated, and the organicphase was washed with 0.625 M aq. NaHCO₃ (80 mL). The combinedbicarbonate extracts were then washed with ethyl acetate (100 mL). Theaqueous phase was then stirred with ethyl acetate (300 mL) and acidifiedwith 12 N HCl (approximately 37 mL). The layers were separated and theacidic aqueous phase was extracted with ethyl acetate (100 mL). Thecombined ethyl acetate extracts were concentrated in vacuo. The residuewas slurried with a minimal amount of ethyl acetate, filtered, washedagain with ethyl acetate and dried to give 50.1 g (73%) of white powder.

¹ H NMR FAB-MS, m/e 369 (M⁺) Analysis for C₁₉ H₃₂ N₂ O₅ :

Calc: C, 61.93; H, 8.75; N, 7.60;

Found: C, 62.01; H, 8.96; N, 7.75.

B) Preparation of 2-fluoroterephthalonitrile

A solution of 4-bromo-2-fluorobenzonitrile (20 g, 100 mmol) , zinccyanide (7 g, 60 mmol) and tetrakis (triphenylphosphine) palladium (4.6g, 4 mmol) in DMF (100 mL) was heated at 80° C. for 4 hr. Toluene (300mL) and saturated aqueous ammonium chloride (300 mL) were added and thelayers were separated. The organic layer was washed once with saturatedaqueous ammonium chloride and twice with brine. The organic phase wasdried (MgSO₄), filtered and concentrated. The product was purified bysilica gel chromatography, eluting with a gradient of hexanes to 30%EtOAc/hexanes (11 g, 75%).

IR ¹ H NMR FD-MS, m/e 146 (M⁺) Analysis for C₈ H₃ FN₂ :

Calc: C, 65.76; H, 2.07; N, 19.17;

Found: C, 65.69; H, 2.33; N, 19.05.

C) Preparation of 3-amino-1,2-benzisoxazole-5-carbonitrile

To a stirring solution of potassium t-butoxide (8.4 g, 75 mmol) in THF(100 mL) was added acetone oxime (5.5 g, 75 mmol). After stirring for 30min, a solution of 2-fluoroterephthalonitrile (10 g, 68 mmol) in THF (50mL) was added; and stirring was continued for an additional 2 hr.Saturated aqueous ammonium chloride (100 mL) was added and the solventswere removed in vacuo. The residue was partitioned between EtOAc andbrine. The layers were separated and the organic phase was washed oncewith brine, dried (MgSO₄), filtered and concentrated. This crude solidwas suspended in a solution of EtOH (150 mL), concentrated HCl (50 mL)and water (100 mL). This mixture was refluxed for 2 h. After cooling toroom temperature, the solvents were removed in vacuo. The residue wastreated with saturated aqueous sodium bicarbonate (200 mL), and theproduct was extracted by washing the aqueous layer three times withEtOAc. This organic solution was washed once with brine, dried (MgSO₄),filtered and concentrated to give a pink colored solid (9.4 g, 86%).

IR ¹ H NMR FD-MS, m/e 159 (M⁺) Analysis for C₈ H₅ N₃ O:

Calc: C, 60.38; H, 3.17; N, 26.40;

Found: C, 60.96; H, 3.44; N, 25.58.

D) Preparation of 4-aminomethyl-2-hydroxybenzamidine dihydrochloride

3-Amino-1,2-benzisoxazole-5-carbonitrile (5 g, 31 mmol) was dissolved inEtOH (130 mL). 5% Pd/C (2.5 g) and 5 N HCl (15 mL) were added and themixture was hydrogenated at 4.1 bar on a shaker for 4 h. The catalystwas filtered, and the filtrate was concentrated to give a tan solid.This was titurated with diethyl ether and collected by filtration (3.2g, 43%).

IR ¹ H NMR FD-MS, m/e 165 (M⁺) Analysis for C₈ H₁₁ N₃ O·2HCl:

Calc: C, 40.35; H, 5.50; N, 17.65; Cl, 29.78;

Found: C, 40.75; H, 6.13; N, 15.91; Cl, 28.26.

E) Preparation of D-Cha-Pro-NHCH₂ C₆ H₃ -3-OH-4-C(NH)NH₂ ·2HCl

To a stirring solution of Boc-D-Cha-Pro-OH (1.1 g, 2.9 mmol),4-aminomethyl-2-hydroxybenzamidine dihydrochloride (0.71 g, 3 mmol) anddiisopropylethylamine (1.7 mL, 10 mmol) in DMF (60 mL) was addedbenzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate (1.6g, 3.1 mmol). After stirring overnight, the solvent was removed invacuo. The resulting residue was partitioned between EtOAc and saturatedaqueous ammonium chloride. The layers were separated and the organicphase was washed once with saturated aqueous ammonium chloride and twicewith brine, dried (MgSO₄), filtered, and concentrated to give a residue.To this was added anisole (2.5 mL) and TFA. (50 mL). The solution wasstirred at room temp for 30 min, followed by removal of TFA in vacuo.The residue was dissolved in 1 N HCl (50 mL) and washed twice withEtOAc. The crude product was concentrated in vacuo and purified by HPLCMethod 1 using a gradient of 98/2 A/B to 50/50 A/B over 2.5 hr.Fractions containing only desired product (as judged by analytical HPLC)were pooled, concentrated and lyophilized to give a white powder (486mg, 35%).

¹ H NMR FAB-MS, m/e 416.3 (MH⁺) Analysis for C₂₂ H₃₃ N₅ O₃ ·2HCl:

Calc: C, 54.10; H, 7.22; N, 14.34;

Found: C, 53.89; H, 7.28; N, 14.07.

Example 2

Preparation ofD-cyclohexylalanyl-N-[[4-(aminoiminomethyl)-3-hydroxy-2,5,6-trifluorophenyl]methyl]-L-prolinamidedihydrochloride ##STR23## A) Preparation of3-amino-4,6,7-trifluoro-1,2-benzisoxazole-5-carbonitrile

To a stirring solution of N,N-diisopropylethylamine (19.2 g, 110 mmol)in CH₃ CN (100 mL) was added acetone oxime (8 g, 110 mmol). Afterstirring for 30 min, a solution of tetrafluoroterephthalonitrile (20 g,100 mmol) in CH₃ CN (50 mL) was added; and the mixture was stirredovernight. Saturated aqueous ammonium chloride (100 mL) was added andthe solvents were removed in vacuo. The residue was partitioned betweenEtOAc and brine. The layers were separated and the organic phase waswashed once with brine, dried (MgSO₄), filtered and concentrated. Thiscrude solid was suspended in EtOH (150 mL) and concentrated HCl (50 mL)and water (100 mL) were added. This was refluxed for 3 h. After coolingto room temperature, the solvents were removed in vacuo. The residue wastreated with saturated aqueous sodium bicarbonate (200 mL), and theproduct was extracted by washing the aqueous layer three times withEtOAc. This organic solution was washed once with brine, dried (MgSO₄),filtered and concentrated to give a yellow solid. The crude product waspurified by silica gel chromatography, eluting with a gradient ofhexanes to 40% EtOAc/hexanes (9.2 g, 43%).

IR ¹ H NMR FD-MS, m/e 213 (M⁺) Analysis for C₈ H₂ F₃ N₃ O:

Calc: C, 45.09; H, 0.95; N, 19.72; F, 26.74;

Found: C, 45.47; H, 1.12; N, 19.39; F, 27.68.

B) Preparation of 4-aminomethyl-2-hydroxy-3,5,6-trifluorobenzamidinedihydrochloride

3-Amino-4,6,7-trifluoro-1,2-benzisoxazole-5-carbonitrile (5 g, 31 mmol)was dissolved in EtOH (130 mL). 5% Pd/C (2.5 g) and 5 N HCl (15 mL) wereadded and the mixture was hydrogenated at 4.1 bar on a shaker for 4 h.The catalyst was filtered, and the filtrate was concentrated to give awhite foam (7.4 g, 100%).

IR ¹ H NMR FD-MS, m/e 219 (M⁺) Analysis for C₈ H₈ F₃ N₃ O·2HCl:

Calc: C, 32.90; H, 3.45; N, 14.38; Cl, 24.28;

Found: C, 32.80; H, 4.00; N, 12.75; Cl, 22.22.

C) Preparation of D-Cha-Pro-NHCH₂ C₆ F₃ -3-OH-4-C(NH)NH₂ ·2HCl

By methods substantially equivalent to those described in example 1-E,0.04 g of D-Cha-Pro-NHCH₂ C₆ F₃ -3-OH-4-C(NH)NH₂ ·2HCl was prepared from4-aminomethyl-2-hydroxy-3,5,6-trifluorobenzamidine dihydrochloride.

¹ H NMR FD-MS, m/e 470 (MH⁺) Analysis for C₂₂ H₃₀ F₃ N₅ O₃ ·2HCl:

Calc: C, 48.71; H, 5.95; N, 12.91;

Found: C, 48.90; H, 6.03; N, 12.86.

Example 3

Preparation ofN-carboxymethyl-D-cyclohexylalanyl-N-[[4-(aminoiminomethyl)-3-hydroxypheny]methyl]-L-prolinamide hydrochloride ##STR24## A)Preparation of N-(t-BuO₂ CCH₂)-N-Boc-D-Cha-Pro-OH

To a solution of D-Phe-Pro-OBn·HCl (20 g, 51 mmol) in DMF (100 mL) wasadded t-butyl bromoacetate (9.9 g, 56 mmol) in one portion andN,N-diisopropylethylamine (17.4 mL, 101 mmol) dropwise over 30 min. Thismixture was allowed to stir for 18 h. Di-t-butyl dicarbonate (16.6 g, 76mmol) and N,N-diisopropylethylamine (13.2 mL, 76 mmol) were then addedin one portion, and the reaction was allowed to stir an additional 24 h.The solvent was removed in vacuo and the residue was partitioned betweenEtOAc (1 L) and 1 M aqueous citric acid (500 mL). The layers wereseparated and the organic phase was washed once with 1 M aqueous citricacid, twice with saturated aqueous sodium bicarbonate, and once withbrine (500 mL each). The organic phase was dried (Na₂ SO₄), filtered,and concentrated in vacuo. The amber oil was purified by silica gelchromatography eluting with a EtOAc/hexanes gradient (hexanes to 30%EtOAc/hexanes). Fractions containing product were combined andconcentrated to give 19.0 g (66%) of N-(t-BuOOCCH₂)-N-Boc-D-Phe-Pro-OBnas a colorless oil which slowly crystallized upon standing.

¹ H NMR FD-MS, m/e 566 (M⁺) Analysis for C₃₂ H₄₂ N₂ O_(:)

Calc: C, 67.82; H, 7.47; N, 4.94;

Found: C, 68.06; H, 7.33; N, 5.17.

To a solution of N-(t-BuOOCCH₂)-N-Boc-D-Phe-Pro-OBn (18.5 g, 33 mmol) inEtOAc (250 mL) was added 5% Pd/C catalyst (5 g). This solution wasdegassed in vacuo several times and placed under an atmosphere ofhydrogen for 2 h with stirring. The balloon was removed, diatomaceousearth was added and the slurry was filtered over a pad of diatomaceousearth. The filtrate was concentrated in vacuo to give 13.2 g (84%) ofN-(t-BuO₂ CCH₂)-N-Boc-D-Phe-Pro-OH as a white foam.

¹ H NMR FD-MS, m/e 476 (M⁺) Analysis for C₂₅ H₃₆ N₂ O₇ :

Calc: C, 63.01; H, 7.61; N, 5.88;

Found: C, 63.23; H, 7.73; N, 5.59.

N-(t-BuO₂ CCH₂)-N-Boc-D-Phe-Pro-OH (13 g, 27 mmol) was dissolved inethanol (750 mL) and PtO₂ (13 g) was added. The suspension was shakenunder an atmosphere of hydrogen (4.1 bar) at 40° C. for 16 h. Thecatalyst was then filtered, and the filtrate was concentrated in vacuoto give 11.7 g (90%) of N-(t-BuO₂ CCH₂)-N-Boc-D-Cha-Pro-OH as a whitefoam.

IR ¹ H NMR FD-MS, m/e 483 (MN⁻) Analysis for C₂₅ H₄₂ N₂ O₇ :

Calc: C, 62.22; H, 8.77; N, 5.80;

Found: C, 2.99; H, 8.96; N, 5.48.

B) Preparation of HO₂ CCH₂ -D-Cha-Pro-NHCH₂ C₆ H₃ -3-OH-4-C(NH)NH₂ ·HCl

By methods substantially equivalent to those described in 1-E, 0.22 g ofHO₂ CCH₂ -D-Cha-Pro-NHCH₂ C₆ H₃ -3-OH-4-C(NH)NH₂ ·HCl was prepared.

¹ H NMR FD-MS, m/e 474.3 (MH⁺) Analysis for C₂₄ H₃₅ N₅ O₅ ·1.5 HCl:

Calc: C, 54.57; H, 6.96; N, 13.26;

Found: C, 54.52; H, 6.95; N, 13.09.

Example 4

Preparation of N-[[4-(aminoiminomethyl)-3-hydroxyphenyl]-methyl]-1-[(1R, 4 aR, 8 aR) -perhydroisoquinolin-1-ylcarbonyl]-L-prolinamidedihydrochloride ##STR25##

1-[(1 R,4 aR,8 aR)-2-Cbz-Perhydroisoquinolin-1-ylcarbonyl]-L-proline ({α}_(D) =34.2° (C=0.5 MeOH)) was obtained as described in U.S. Pat. No.5,430,023 at Example 25, column 23, line 23 through column 24, line 46.This compound also is known as Cbz-D-cis[4 aR,8 aR]-1-Piq-Pro-OH.

To a stirring solution of Cbz-(1 R,4 aR,8 aR)-1-Piq-Pro-OH (1.1 g, 2.5mmol), 4-aminomethyl-2-hydroxybenzamidine dihydrochloride (0.66 g, 2.75mmol) and diisopropylethylamine (1.5 mL, 8.8 mmol) in DMF (60 mL) wasadded benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate(1.4 g, 2.75 mmol). After stirring overnight, the solvent was removed invacuo. The resulting residue was partitioned between EtOAc and saturatedaqueous ammonium chloride. The layers were separated and the organicphase was washed once with saturated aqueous ammonium chloride and twicewith brine, dried (MgSO₄), filtered, and concentrated to give a residue.This residue was dissolved in EtOH (10 mL) and water (50 mL). 1 N HCl (5mL) and 5% Pd/C (0.5 g) were added. The slurry was degassed and themixture placed under a hydrogen atmosphere overnight. Diatomaceous earthwas added and the slurry was filtered over a pad of diatomaceous earthand concentrated in vacuo. The crude product was purified by HPLC Method1 using a gradient of 98/2 A/B to 30/70 A/B over 2.5 hr. Fractionscontaining pure product (as judged by analytical HPLC) were pooled,concentrated and lyophilized to give a white powder (0.22 g, 18%).

¹ H NMR FAB-MS, m/e 428.3 (MH⁺) Analysis for C₂₃ H₃₃ N₅ O₃ -2HCl:

Calc: C, 55.20; H, 7.05; N, 13.99;

Found: C, 54.93; H, 7.31; N, 14.00.

Example 5

Preparation ofN-ethylsulfonyl-D-cyclohexylalanyl-N-[[4-(aminoiminomethyl)-3-hydroxyphenyl]methyl]-L-prolinamidehydrochloride ##STR26## A) Preparation of EtSO₂ -D-Phe-OH

To a stirring suspension of D-phenylalanine (50 g, 300 mmol) in THF (400mL) was added N,O-bis(trimethylsilyl) acetamide (92 g, 450 mmol). Afterstirring for 12 h, the solution was cooled to -78° C. andN,N-diisopropylethylamine (58 mL, 330 mmol) was added. To this solutionwas slowly added ethanesulfonyl chloride (31 mL, 330 mmol) and the coldbath was removed. After stirring for 20 h, the solvents were removed invacuo and the residue was partitioned between saturated aqueous NaHCO₃and ethyl acetate. The aqueous phase was washed with diethyl ether,acidified with solid citric acid and extracted twice with ethyl acetate.The combined ethyl acetate extracts were washed with brine, dried withMgSO₄, filtered and concentrated in vacuo to give 61 g (79%) of a thickcolorless oil.

IR ¹ H-NMR FD-MS, m/e 257 (M⁺)

B) Preparation of EtSO₂ -D-Phe-Pro-OBn

To a stirring suspension of EtSO₂ -D-Phe-OH (25.7 g, 100 mmol),Pro-OBn·HCl (26.6 g, 110 mmol), HOBT (13.5 g, 100 mmol) andN,N-diisopropylethylamine (43.5 mL, 250 mL) in THF (1 L) at 0° C. wasadded 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (23 g,120 mL). After stirring for 20 h, the solvent was removed in vacuo andthe residue was partitioned between ethyl acetate and 1 N citric acid.The organic phase was washed twice with 1 N KHCO₃, twice with brine,dried with MgSO₄, filtered and concentrated in vacuo. The residue waschromatographed over silica gel, eluting with a step gradient of hexanesthrough 50% ethyl acetate/hexanes. The product containing fractions werecombined and concentrated in vacuo to give 29 g (65%) of a clear, thickoil.

IR ¹ H-NMR FD-MS, m/e 444 (M⁺)

C) Preparation of EtSO₂ -D-Phe-Pro-OH

To a solution of EtSO₂ -D-Phe-Pro-OBn (28.5 g, 64 mmol) in ethyl acetate(500 mL) was added 10% Pd/C (5 g). The vessel was evacuated and placedunder an atmosphere of hydrogen. After stirring for 16 h, the solutionwas filtered over diatomaceous earth, and the filter pad was then washedtwice with methanol and filtered. The combined filtrates wereconcentrated in vacuo to give 22 g (97%) of off-white solid.

IR ¹ H-NMR FD-MS, m/e 355 (MH⁺) Analysis for C₁₆ H₂₂ N₂ O₅ S:

Calc: C, 54.22; H, 6.26; N, 7.90;

Found: C, 53.98; H, 6.12; N, 7.63.

D) Preparation of EtSO₂ -D-Cha-Pro-OH

To a solution of EtSO₂ -D-Phe-Pro-OH (10 g, 28 mmol) in ethanol (300 mL)was added PtO₂ (5 g). The mixture was hydrogenated using a high pressureapparatus at 4.1 bar and 20° C. for 20 h. The solution was then filteredthrough diatomaceous earth, and concentrated to give 8.1 g (80%) ofthick oil.

IR ¹ H-NMR FD-MS, m/e 361 (MH⁺)

E) Preparation of EtSO₂ -D-Cha-Pro-NHCH₂ C₆ H₃ -3-OH-4-C (NH)NH₂ ·2HCl

To a stirring solution of EtSO₂ -D-Cha-Pro-OH (0.87 g, 2.4 mmol),4-aminomethyl-2-hydroxybenzamidine dihydrochloride (0.63 g, 2.64 mmol)and diisopropylethylamine (1.5 mL, 8.8 mmol) in DMF (60 mL) was addedbenzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (1.4g, 2.75 mmol). After stirring overnight, the solvent was removed invacuo. The residue was partitioned between 1 N HCl and Et₂ O. The layerswere separated and the aqueous phase was washed three times with Et₂ Oand concentrated in vacuo. The crude product was purified by HPLC Method1 using a gradient of 90/10 A/B to 40/60 A/B over 2.5 hr. Fractionscontaining pure product (as judged by analytical HPLC) were pooled,concentrated and lyophilized to give a white powder (0.30 g, 21%).

¹ H NMR FD-MS, m/e 508 (MH⁺) Analysis for C₂₄ H₃₇ N₅ O₅ S·3HCl:

Calc: C, 46.72; H, 6.53; N, 11.35;

Found: C, 46.36; H, 6.16; N, 11.22.

Example 6

Preparation ofN-ethylsulfonyl-D-cyclohexylalanyl-N-[[4-(aminoiminomethyl)-3-hydroxy-2,5,6-trifluorophenyl]methyl]-L-prolinamidehydrochloride ##STR27##

By a method substantially equivalent to that described in Example 5,0.54 g of EtSO₂ -D-Cha-Pro-NHCH₂ C₆ F₃ -3-OH-4-C (NH)NH₂ ·HCl wasprepared starting from EtSO₂ -D-Cha-Pro-OH and4-aminomethyl-2-hydroxy-3,5,6-trifluorobenzamidine dihydrochloride.

¹ H NMR FAB-MS, m/e 562.2 (MH⁺) Analysis for C₂₃ H₃₄ F₃ N₅ O₅ S·2HCl:

Calc: C, 45.43; H, 5.72; N, 11.04;

Found: C, 45.54; H, 5.61; N, 11.03.

Example 7

Preparation of1-[N-ethylsulfonyl-D-phenylalanyl]-N-[[4-(aminoiminomethyl)-3-hydroxyphenyl]methyl]-[2S-(2α,3αβ,7αβ)]-octahydroindole-2-carboxamidehydrochloride ##STR28## A) Preparation of[2S-(2α,3αβ,7αβ)]-octahydroindole-2-carboxylic acid ethyl ester·HCl(Ohi-OEt·HCl)

HCl gas was bubbled through a stirring suspension of(S)-indoline-2-carboxylic acid (20 g, 110 mmol) in ethanol (400 mL).When the acid was completely dissolved, the solution was brought toreflux. After 16 hours, the solution was cooled and the solvent removedin vacuo. The residue was triturated with diethyl ether and theresulting off-white solid was collected by filtration, washed withhexanes and dried overnight in a vacuum oven at 30° C. (25.5 g, 100%).This solid, (S)-indoline-2-carboxylic acid ethyl ester hydrochloride,was dissolved in ethanol (455 mL). To this was added 5% Pd/C (25.5 g)and the resulting suspension was hydrogenated at 4.1 bar on a shaker for8 hours. The solution was filtered to remove catalyst and the filtratewas concentrated in vacuo. The residue was triturated with diethyl etherand the resulting solid was isolated by filtration to give 18.8 g (73%)of a white powder.

¹ H NMR FD-MS, m/e 197 (M⁺) Analysis for C₁₁ H₁₉ NO₂ ·HCl:

Calc: C, 56.53; H, 8.63; N, 5.99;

Found: C, 56.24; H, 8.44; N, 6.00.

B) EtSO₂ -D-Phe-Ohi-OEt

By methods substantially equivalent to those described in example 5,EtSO₂ -D-Phe-Ohi-OEt was prepared (57%) from EtSO₂ -D-Phe-OH andHCl·Ohi-OEt.

IR ¹ H NMR FD-MS, m/e 436.1 (M⁺) Analysis for C₂₂ H₃₂ N₂ O₅ S:

Calc: C, 60.53; H, 7.39; N, 6.42;

Found: C, 60.62; H, 7.31; N, 6.22.

C) EtSO₂ -D-Phe-Ohi-OH

To a stirring solution of EtSO₂ -D-Phe-Ohi-OEt (12 g, 27.5 mmol) inp-dioxane (300 mL) was added a solution of LiOH·H₂ O (2.3 g, 55 mmol) inwater (150 mL). After stirring for 16 h, the solvent was removed invacuo and the residue was redissolved in water and washed twice withdiethyl ether. The aqueous phase was acidified with 5 N HCl and theprecipitate was filtered, washed with water and dried in vacuo to give10.1 g (90%) of a light yellow solid.

IR ¹ H NMR FD-MS, m/e 409.1 (M⁺) Analysis for C₂₀ H₂₈ N₁ O₅ S:

Calc: C, 58.80; H, 6.91; N, 6.86;

Found: C, 58.57; H, 7.00; N, 6.63.

D) Preparation of EtSO₂ -D-Phe-Ohi-NHCH₂ C₆ H₃ -3-OH-4-C (NH)NH₂ ·HCl

By methods substantially equivalent those described in Example 5, 400 mgof EtSO₂ -D-Phe-Ohi-NHCH₂ C₆ H₃ -3-OH-4-C (NH)NH₂ ·HCl was obtained.HPLC Method 1 (gradient of 80/20 A/B to 30/70 A/B over 2.5 hr) was used.

¹ H NMR FD-MS, m/e 556.1 (MH⁺) Analysis for C₂₈ H₃₇ N₅ O₅ S·2HCl·1.3H₂O:

Calc: C, 51.58; H, 6.43; N, 10.74;

Found: C, 51.51; H, 6.22; N, 10.71.

Example 8

Preparation of1-[N-ethylsulfonyl-D-cyclohexylalanyl]-N-[[4-(aminoiminomethyl)-3-hydroxyphenyl]methyl]-[2S-(2α,3αβ,7αβ)]-octahydroindole-2-carboxamidehydrochloride ##STR29## A) Preparation of EtSO₂ -D-Cha-Ohi-OH

By methods substantially equivalent to those described in example 5-D,EtSO₂ -D-Cha-Ohi-OH was prepared (95%) from EtSO₂ -D-Phe-Ohi-OH.

IR FD-MS, m/e 415.3 (MH⁺)

B) Preparation of EtSO₂ -D-Cha-Ohi-NHCH₂ C₆ H₃ -3-OH-4-C (NH)NH₂ ·HCl

By methods substantially equivalent to those described in Example 5-E,744 mg was obtained.

¹ H NMR FD-MS, m/e 562.1 (MH⁺) Analysis for C₂₈ H₄₃ N₅ O₅ S·2HCl·2H₂ O:

Calc: C, 50.15; H, 7.36; N, 10.44;

Found: C, 50.31; H, 6.97; N, 10.49.

Example 9

Preparation of1-(N-ethylsulfonyl-D-phenylalanyl]-N-[[4-(aminoiminomethyl)-3-hydroxyphenyl]methyl]-S-azetidine-2-carboxamidehydrochloride ##STR30##

By methods substantially equivalent to those described in Example 5, 350mg was obtained.

¹ H NMR FD-MS, m/e 488.0 (MH⁺) Analysis for C₂₃ H₂₉ N₅ O₅ S·3HCl:

Calc: C, 46.28; H, 5.40; N, 11.73;

Found: C, 46.22; H, 5.10; N, 11.49.

Example 10

Preparation of1-[N-ethylsulfonyl-D-cyclohexylalanyl]-N-[[4-(aminoiminomethyl)-3-hydroxyphenyl]methyl]-S-azetidine-2-carboxamidehydrochloride ##STR31##

By methods substantially equivalent to those described in Example 5, 284mg was obtained.

¹ H NMR FD-MS, m/e 494.0 (MH⁺) Analysis for C₂₃ H₃₅ N₅ O₅ S·2.5HCl·0.9H₂O:

Calc: C, 45.97; H, 6.59; N, 11.65;

Found: C, 46.25; H, 6.27; N, 11.31.

Example 11

Preparation ofN-ethylsulfonyl-D-phenylalanyl-N-[[4-(aminoiminomethyl)-3-hydroxyphenyl]methyl]-L-prolinamidehydrochloride ##STR32##

By methods substantially equivalent to those described in Example 5, 151mg was obtained.

¹ H NMR FD-MS, m/e 502.1 (MH⁺) Analysis for C₂₄ H₃₁ N₅ O₅ S·3HCl·1.5H₂O:

Calc: C, 45.18; H, 5.85; N, 10.97;

Found: C, 45.12; H, 5.45; N, 10.85.

Example 12

Preparation ofN-ethylsulfonyl-D-phenylalanyl-N-[[4-(aminoiminomethyl)-3-hydroxyphenyl]methyl]sarcosinamidehydrochloride ##STR33##

By methods substantially equivalent to those described in Example 5, 151mg was obtained.

¹ H NMR FD-MS, m/e 476.1 (MH⁺) Analysis for C₂₂ H₂₉ N₅ O₅ S·2.5HCl·0.5H₂O:

Calc: C, 45.90; H, 5.69; N, 12.16;

Found: C, 45.72; H, 5.36; N, 12.03.

What is claimed is:
 1. An amine of Formula III

    H.sub.2 N--(CH.sub.2).sub.s --R                            III

wherein s is 1 or 2 and R is a 4-amidino-3-hydroxyphenyl group bearing0, 1, 2 or 3 fouoro substituents.
 2. An amine as claimed in claim 1 ofFormula IIIa ##STR34## in which f is 0, 1, 2 or
 3. 3. A compound asclaimed in claim 2 in which f is 0 or
 3. 4. A compound as claimed inclaim 3 in which f is
 0. 5. A compound as claimed in claim 3 in which fis 3.